Journal of Applied Microbiology 1998, 85, 657–663

Use of bacteriocinogenic lactic acid bacteria to inhibit

spontaneous nisin-resistant mutants of Listeria monocytogenes
Scott A
U. Schillinger, H. -S. Chung, K. Keppler and W. H. Holzapfel
Institut für Hygiene und Toxikologie, Bundesforschungsanstalt für Ernährung, Karlsruhe, Germany
6591/02/98: received 9 February 1998, revised 17 April 1998 and accepted 28 April 1998

Nisin is a bacteriocin
U . S CH I LL IN G ER , H . -S . C H UN G, K . K EP P LE R A N D W .H . HO LZ A PF EL . 1998.
with a broad antibacterial spectrum including strains of Listeria monocytogenes. Populations
of L. monocytogenes, however, frequently contain spontaneous nisin-resistant mutants. When
a culture of L. monocytogenes Scott A was exposed to nisin concentrations between 10 and
500 IU ml−1, the initial decrease in viable numbers was followed by regrowth of survivors to
nisin. Nisin-resistant mutants of L. monocytogenes Scott A were isolated after a single exposure
to nisin at 100 IU ml−1 and were shown to be sensitive to the non-nisin bacteriocins, sakacin
A and enterocin B, produced by Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900,
respectively. The regrowth of L. monocytogenes Scott A following the initial decrease due to
exposure to nisin was prevented by nisin-resistant Lact. sake Lb 706–1a and to a somewhat
lesser extent, by Ent. faecium BFE 900–6a. Listerial cells surviving nisin action were thus
inhibited by the bacteriocin-producing strains that might be used as starter or protective
cultures in foods. Growth of a nisin-resistant mutant of L. monocytogenes Scott A (Li3) was
also suppressed by the bacteriocinogenic cultures. Use of nisin in combination with a starter
culture producing a non-nisin antilisterial bacteriocin may therefore prevent the emergence
of nisin-resistant mutants of L. monocytogenes.

INTRODUCTION resistant population. Therefore it cannot be excluded that

Nisin is widely used as a preservative in several food products
such as processed cheese, dairy desserts and canned foods. It
shows inhibitory activity against spore-forming bacteria and
other Gram-positive spoilage and pathogenic bacteria includ-
ing Listeria monocytogenes. Among the sensitive bacteria, how-
ever, strains differ greatly in their relative sensitivity to nisin.
Some strains of L. monocytogenes may be inhibited by low
concentrations of nisin (below 200 IU ml−1) whereas others
are relatively resistant and require much higher amounts of
nisin to be inhibited (Ferreira and Lund 1996; Ukuku and
Shelef 1997). Even within a population of sensitive bacteria,
some cells show elevated resistance to nisin. Spontaneous
nisin-resistant variants of L. monocytogenes are reported to
occur at a relatively high frequency (Harris et al. 1991; Ming
and Daeschel 1993; Davies and Adams 1994). Even a single
exposure to nisin may result in the emergence of a nisin-
Correspondence to: Ulrich Schillinger, Institut für Hygiene und Toxikologie,
Bundesforschungsanstalt für Ernährung, Engesserstr.20, D-76131 Karlsruhe,
Germany (e-mail: Ulrich.schillinger@bfe.uni-karlsruhe.de).
© 1998 The Society for Applied Microbiology
nisin-resistant variants of L. monocytogenes arise from wide-
spread application of nisin in foods. On the other hand, the
resistance problem may be minimized by the use of nisin in
combination with other food preservatives or preservation
techniques. Low pH, increased salt concentration or modified
atmosphere packaging are examples of additional ‘hurdles’ in
foods to be overcome by the target bacteria. The application
of nisin in combination with such preservation measures may
reduce the risk of emergence of nisin-resistant populations of
L. monocytogenes.
Another approach could be the use of starter or protective
cultures producing antilisterial bacteriocins active against
nisin-resistant cells of L. monocytogenes. Numerous studies
have shown that bacteriocin-producing lactic acid bacteria
are able to suppress growth of L. monocytogenes in meat and
other foods (Schillinger et al. 1991; Foegeding et al. 1992;
Degnan et al. 1992; Maisnier-Patin et al. 1992; Winkowski
et al. 1993; Stiles 1996). There are, however, no studies on
the effectiveness of bacteriocinogenic cultures towards nisin-
resistant mutants of L. monocytogenes.
658 U . S CH I LL IN G ER ET A L.
The objective of this study was to investigate whether the
emergence of nisin-resistant mutants of L. monocytogenes
Scott A can be prevented by the addition of a bacterial strain
producing a non-nisin antilisterial bacteriocin.
MATERIALS AND METHODS
Nisin
Nisaplin, a commercial product of nisin that contained 106
IU g−1, was provided by Aplin & Barret Ltd (Trowbridge,
UK). Stock solutions of nisin were prepared by dissolving
100 mg of Nisaplin in 10 ml of 0·02 N HCl. The pH was
adjusted to 2·0 with 1 N NaOH. The solutions were subjected
to heating at 100 °C for 10 min and then stored at – 20 °C.
Bacterial strains and culture media
Lactobacillus sake Lb 706 (Schillinger and Lücke 1989) and
Enterococcus faecium BFE 900 (Franz et al. 1996) were used
as bacteriocinogenic protective cultures and L. monocytogenes
Scott A as target organism. In some experiments, a bac-
teriocin-negative mutant of Lact. sake Lb 706 (Lb 706-B)
was included (Schillinger and Lücke 1989). Lactobacillus sake
DSM 20017 was used as indicator strain for the determination
of bacteriocin activity. Listeria monocytogenes was grown in
Standard I Nutrient broth (Merck) and the lactic acid bacteria
were cultivated in MRS broth (Merck) at 30 °C. The cultures
were maintained as frozen stocks at – 20 °C in 15% glycerol.
Preparation of sakacin A and enterocin B and
bacteriocin assay
Partial purification of the bacteriocins sakacin A from Lact.
sake Lb 706 and enterocin B from Ent. faecium BFE 900 was
performed as described by Keppler et al. (1994) for carnocin
54. In the present purification protocol, Amberlite XAD-7
(Fluka, Switzerland) was used instead of Amberlite XAD-2
and the concentrated bacteriocin solution obtained after
rotary evaporation was adjusted to pH 5·0 with ammonia.
Bacteriocin activity was tested using the agar spot assay
described previously (Schillinger et al. 1993).
Determination of Minimum Inhibitory Concentrations
(MIC)
The minimum inhibitory concentration (MIC) of nisin, sak-
acin A and enterocin B was determined for L. monocytogenes
Scott A and mutants derived from this strain. Serial 1:1
dilutions of the respective bacteriocin were prepared using
Standard I Nutrient broth in microtitre plates and each well
was inoculated with about 106 cells ml−1. The MIC was
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 657–663
recorded as the lowest concentration of the bacteriocin which
prevented visible growth after incubation for 20 h at 30 °C.
Sensitivity of Lactobacillus sake Lb 706 and
Enterococcus faecium BFE 900 to nisin and generation
of nisin-resistant derivatives
Sensitivity of Lact. sake Lb 706 and Ent. faecium BFE 900
to nisin was determined by growing them in MRS broth
containing various concentrations of nisin for 20 h at 30 °C.
In order to generate nisin-resistant mutants, Lact. sake Lb
706 and Ent. faecium BFE 900 were inoculated at 1% in MRS
broth. Following incubation for 24 h at 30 °C, cultures were
serially transferred to MRS broth containing increasing con-
centrations of nisin in 100 IU ml−1 steps. Transfers were
carried out every 24 or 48 h or as soon as turbidity indicated
growth. Turbid growth cultures with 500 IU of nisin ml−1
were streaked onto MRS agar plates with 500 IU of nisin
added. Colonies were isolated and checked for nisin resistance
by determining the MIC. Bacteriocin production was tested
by using culture supernatant fluids in the agar spot assay.
Alternatively, nisin-resistant mutants were obtained by
direct streaking of overnight cultures onto MRS agar plates
containing 300 IU of nisin. Several colonies isolated from
these plates were transferred to MRS broth containing 300–
500 IU of nisin ml−1, and after incubation at 30 °C for 24–
48 h, they were checked for nisin resistance (300 IU ml−1)
and bacteriocin production.
SDS PAGE electrophoresis of whole cell proteins of those
isolates was done to confirm the identity of the derivative and
parental strains (Pot et al. 1994).
Stability of the nisin-resistance phenotype
The stability of the nisin-resistant derivatives of Lact. sake Lb
706 and Ent. faecium BFE 900 was determined as described by
Breidt et al. (1993). Nisin-resistant cultures of Lact. sake Lb
706 and Ent. faecium BFE 900 were transferred daily for 14 d
in MRS broth in the absence of nisin, with a 1% inoculum
at each transfer. Samples were withdrawn every second day
and frozen at – 20 °C in MRS broth containing 15% glycerol.
At the end of this 14 day period, growth of each sample in
the presence of nisin was tested using an automated tur-
bidometric system, BIOSCREEN C (Labsystems, Helsinki,
Finland) according to Holck et al. (1996). Honeycomb wells
with MRS broth containing 300 IU of nisin ml−1 were inocu-
lated with the cultures at 1% and were incubated for 24 h at
30 °C. The non-resistant parental cultures and MRS broth
without nisin were included as controls.
Bactericidal effect of nisin against Listeria
monocytogenes Scott A
Standard I Nutrient broth was supplemented with nisin at a
concentration of 10, 50, 100 and 500 IU ml−1 and was inocu-

lated with about 108 viable cells of L. monocytogenes Scott A
ml−1. Numbers of survivors were determined after incubation
for 2 h and 24 h at 30 °C.
Isolation of nisin-resistant mutants of Listeria
monocytogenes Scott A
Listeria monocytogenes Scott A (105 cfu) were inoculated into
1 ml Standard I Nutrient broth containing 100 IU of nisin.
After incubation for 3 h at 30 °C, diluted samples were plated
on Standard I Nutrient agar plates. Seven colonies were
isolated from these plates, transferred into nisin-free broth
and re-tested for nisin susceptibility by determining the MIC.
To test stability of the nisin resistance phenotype of isolate
Li3, the culture was transferred daily for 6 d in nisin-free
Standard I Nutrient broth. Samples from every day were
tested for nisin resistance by determining the MIC value.
Inhibition of Listeria monocytogenes by nisin in
combination with a nisin-resistant derivative of
Lactobacillus sake Lb 706 and Enterococcus faecium
BFE 900
Standard I Nutrient broth, pH 7·5 and with 100 IU of nisin
ml−1 added, was inoculated with L. monocytogenes Scott A or
Li3 at an initial level of either 104 or 106 cfu ml−1, and with
Lact. sake Lb 706–1a or Ent. faecium BFE 900–6a at about
105–106 cfu ml−1. Incubation was at 30 °C for 48 h. Bacterial
viable counts were determined after different time intervals
using MRS agar for Lact. sake and Ent. faecium, and PAL-
CAM agar (Merck) for Listeria. In some experiments, a bac-
teriocin-negative mutant of Lact. sake Lb 706 (Lb 706-B) was
included for comparison, and pH and bacteriocin activity of
the supernatant fluids were determined after different time
intervals by using the critical dilution method (agar spot
assay). All experiments were done in duplicate.
RESULTS
Isolation of nisin-resistant mutants of Listeria
monocytogenes Scott A
Exposure of L. monocytogenes Scott A to increasing con-
centrations of nisin resulted in an initial decrease of viable
counts followed by a resurgence of growth to high cell num-
bers (Fig. 1). Even at the highest nisin concentration of 500
IU ml−1, Listeria numbers did not remain at a low level below
10 cfu ml−1 but increased to 6 × 104 cfu ml−1 within 24 h
and to 108 cfu ml−1 after 48 h. Nisin was not inactivated by
the listerial cells as the culture supernatant fluid was still
active against L. monocytogenes Scott A. Surviving cells of
Listeria were obviously able to grow in the presence of nisin,
indicating resistance to nisin levels of 100–500 IU ml−1. From
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 657–663
I NH IB I TI ON O F N IS I N- RE S IS TA N T L IS T ER IA 659
12
10
Log cfu ml–1
8
6
4
2
0
0 20 40 60 80
Time (h)
Fig. 1 Bactericidal effect of various concentrations of nisin
against Listeria monocytogenes Scott A at 30 °C. (R), 0 IU ml−1;
(ž), 10 IU ml−1; (), 50 IU ml−1; (), 100 IU ml−1; (Ž), 500
IU ml−1
a culture of L. monocytogenes Scott A exposed to 100 IU of
nisin ml−1, seven survivors were isolated and their resistance
to nisin determined. For six of them, a two- to threefold
increased MIC of nisin was found. The seventh strain (Li3)
was four times more resistant to nisin than the parental strain
and was chosen for the further experiments. Several transfers
in media without nisin did not reduce the resistance level of
this strain.
Sensitivity of Listeria monocytogenes Li3 to sakacin A
and enterocin B
Determination of the MIC of sakacin A from Lact. sake
Lb 706 and enterocin B from Ent. faecium BFE 900 for L.
monocytogenes Li3 showed that this nisin-resistant mutant
was still susceptible to both non-nisin bacteriocins (Table 1).
Listeria monocytogenes Scott A and its derivative Li3 did not
differ in their sensitivity to sakacin A and enterocin B. The
finding that L. monocytogenes Li3 had not acquired resistance
to sakacin A is also supported by the observation that inhi-
bition zones of about the same size were produced by Lact.
sake Lb 706 against L. monocytogenes Scott A and Li3 in the
agar spot assay.
Table 1 MIC of Listeria monocytogenes Scott A and Li3 for nisin
(IU ml−1), sakacin A (AU ml−1) and enterocin B (AU ml−1)
—–––––––––––––––––––––––––––––––––––––––––––––––––––––
Bacteriocin L. monocytogenes Scott A L. monocytogenes Li3
—–––––––––––––––––––––––––––––––––––––––––––––––––––––
Nisin 300 1200
Sakacin A 7200 7200
Enterocin B 640 640
—–––––––––––––––––––––––––––––––––––––––––––––––––––––
660 U . S CH I LL IN G ER ET A L.

12
Isolation of nisin-resistant mutants of Lactobacillus (a)
sake and Enterococcus faecium 10

Lactobacillus sake Lb 706 and Ent. faecium BFE 900 were

Log cfu ml–1

8
both sensitive to low nisin concentrations (below 100 IU
6
ml−1). Lactobacillus sake Lb 706 was inhibited even by 10 IU
of nisin ml−1. Nisin-resistant mutants of Lact. sake Lb 706 4
and Ent. faecium BFE 900 were obtained by step-wise
2
exposure to increasing concentrations of nisin. These mutants
were able to grow in the presence of 500 IU of nisin ml−1 0
and were still able to produce the respective bacteriocin. The 0 10 20 30 40 50
stability of the resistance phenotype was determined for three Time (h)
mutants each of Lact. sake Lb 706 and Ent. faecium BFE 900.
10
All of the mutants tested retained the nisin resistance trait (b)
for at least 100 generations in the absence of nisin selection. 8
The mutants did not differ in their whole-cell protein pattern

Log cfu ml–1

obtained by sodium dodecyl sulphate polyacrylamide gel elec- 6
trophoresis (SDS-PAGE) from the original cultures of Lact.
sake Lb 706 and Ent. faecium BFE 900. 4

Inhibition of Listeria monocytogenes by a combination
of nisin and a nisin-resistant culture of Lactobacillus
sake and Enterococcus faecium
When Lact. sake Lb 706–1a, a nisin-resistant mutant of Lact.
sake Lb 706, was added to a culture of L. monocytogenes Scott
A exposed to nisin at a concentration of 100 IU ml−1, the re-
growth of survivors to nisin was inhibited at 30 °C (Fig. 2a).
In the presence of Lact. sake Lb 706–1a, Listeria viable counts
decreased to a level below 100 cfu ml−1, compared with an
increase in Listeria viable numbers to 3 × 108 cfu ml−1 after
48 h observed in the absence of the bacteriocin-producing
Lactobacillus. Similar results were obtained using the nisin-
resistant derivative of Ent. faecium BFE 900 as protective
culture. Enterococcus faecium BFE 900–6a also prevented rest-
oration of listerial cells surviving nisin amounts of 100 IU
ml−1 (Fig. 2b). In the presence of the Enterococcus strain, L.
monocytogenes numbers remained below 104 cfu ml−1
throughout the experiment.
The inhibitory effect of Lact. sake Lb 706–1a and Ent.
faecium BFE 900 was also investigated in combination with
nisin against the nisin-resistant L. monocytogenes Li3. In the
presence of 100 IU of nisin ml−1, L. monocytogenes Li3,
after a short lag phase, grew rapidly to a high cell density
(Fig. 3a,b). This increase in viable numbers was, however,
inhibited by both bacteriocinogenic protective cultures inocu-
lated at a level of 106 cfu ml −1. With Lact. sake Lb 706–1a
added, almost no increase in viable counts of L. monocytogenes
Li3 was oberved after 24 h and the numbers decreased to a
low level after 48 h (Fig. 3a). Enterococcus faecium BFE 900–
6a was somewhat less effective than Lact. sake in controlling
the growth of L. monocytogenes Li3. It did not prevent an
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 657–663
0
0 10 20 30 40 50
Time (h)
Fig. 2 Growth of Listeria monocytogenes Scott A and Lactobacillus
sake Lb 706-1a (a) or Enterococcus faecium BFE 900-6a (b) in mixed
cultures in Standard I Nutrient broth with nisin (100 IU ml−1)
added at 30 °C. (R), L. monocytogenes Scott A alone; (ž), L.
monocytogenes Scott A with Lact. sake Lb 706-1a; (Ž), L.
monocytogenes Scott A with Ent. faecium BFE 900-6a; (),
Lact. sake Lb 706-1a with L. monocytogenes Scott A; (), Ent.
faecium BFE 900-6a with L. monocytogenes Scott A
increase of Listeria viable counts by almost 2 log cycles within
24 h (Fig. 3b). Figure 3(a,b) also shows that viable numbers
of the nisin-sensitive L. monocytogenes Scott A were kept
below 10 cfu ml−1 by Lact. sake Lb 706–1a and Ent. faecium
BFE 900–6a.
Thus, the bacteriocinogenic cultures were effective in sup-
pressing growth of both L. monocytogenes Scott A and the
nisin-resistant mutant Li3.
In a similar experiment, bacteriocin amounts produced by
Lact. sake in a mixed culture with L. monocytogenes Li3 were
determined. As a control, a bacteriocin-negative mutant of
Lact. sake, Lb 706-B, was included. This derivative of Lact.
sake Lb 706 did not produce sakacin A during growth in
mixed culture with L. monocytogenes Li3 in Standard I Nutri-
ent broth, whereas Lact. sake Lb 706–1a released large
amounts of sakacin A into the medium (Fig. 4b). Growth of
L. monocytogenes Li3 was more efficiently suppressed by the
 I NH IB I TI ON O F N IS I N- RE S IS TA N T L IS T ER IA 661

10 10
(a) (a)
8 8

–1
Log cfu ml–1

Log cfu ml
6 6

4 4

2 2

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time (h) Time (h)

10
(b) 9 1200
(b)
8 8 1000

AU × 100 m–1
Log cfu ml–1

6 7
800

pH
6
4 600
5
2 400
4

0 3 0
0 10 20 30 40 50 0 10 20 30 40 50
Time (h) Time (h)

Fig. 3 Growth of Listeria monocytogenes Scott A and its nisin-
resistant derivative Li3 co-cultured with Lactobacillus sake
Lb 706-1a (a) or Enterococcus faecium BFE 900-6a (b) in Standard
I Nutrient broth with nisin (100 IU ml−1) added at 30 °C. (), L.
monocytogenes Scott A without nisin; (ž), L. monocytogenes Scott
A with nisin; (R), L. monocytogenes Scott A with nisin and Lact.
sake Lb 706-1a or Ent. faecium BFE 900-6a; (), L.
monocytogenes Li3 with nisin; (Ž), L. monocytogenes Li3 with
nisin and Lact. sake Lb 706-1a or Ent. faecium BFE 900-6a; (T),
Lact. sake Lb 706-1a or Ent. faecium BFE 900-6a with nisin and L.
monocytogenes Scott A
Fig. 4 Growth of Lactobacillus sake Lb 706-1a (bacteriocin-
positive), Lb 706-B (bacteriocin-negative) and Listeria monocytogenes
Li3 in mixed cultures (a) and bacteriocin production and change
of pH (b) in Standard I Nutrient broth at 30 °C. (ž), Lact.
sake Lb 706-1a with L. monocytogenes Li3; (), Lact. sake Lb
706-B with L. monocytogenes Li3; (), L. monocytogenes Li3
with Lact. sake Lb 706-1a; (Ž), L. monocytogenes Li3 with Lact.
sake Lb 706-B; (R), pH of mixed culture with Lact. sake Lb 706-
1a; (T), pH of mixed culture with Lact. sake Lb 706-B; (e),
bacteriocin activity of Lact. sake Lb 706-1a

Li3 more sensitive to sakacin A produced by Lact. sake Lb

706–1a.

sakacin A producing culture than by the bacteriocin-negative

variant (Lb 706-B) of Lact. sake, indicating that sakacin A
contributed to the inhibition of Listeria (Fig. 4a). The bac-
teriocin-negative mutant of Lact. sake grew to a lower cell
density in Standard I Nutrient broth than Lact. sake Lb 706–
1a. Its growth, however, resulted in the same acidification
effect. With both strains of Lact. sake, pH dropped to the
same level at about the same rate.
Comparison of the growth behaviour of L. monocytogenes
Li3 in mixed culture with Lact. sake with and without nisin
showed that the inhibitory effect of Lact. sake Lb 706–1a
towards Listeria was not more pronounced in the presence of
nisin. About the same reduction in viable counts was caused
by Lact. sake with nisin added as without nisin (Fig. 5).
Exposure to nisin obviously did not render L. monocytogenes
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 657–663
DISCUSSION
Although L. monocytogenes is known to be sensitive to nisin,
populations of Listeria frequently contain spontaneous nisin-
resistant mutants. Harris et al. (1991) detected mutants of L.
monocytogenes resistant to 2000 IU of nisin ml−1 at a frequency
of 10–6–10–8. Ming and Daeschel (1993) observed resistance
frequencies of about 10–6 for L. monocytogenes Scott A when
nisin was added at a level of 400 IU ml−1. The results of our
study indicate that mutants resistant to 100 IU of nisin ml−1
occur at a higher frequency. Exposure of 8 × 103 viable cells
of L. monocytogenes Scott A to 100 IU of nisin did not result
in inactivation of all listerial cells as after an initial decrease
of viable numbers below 10 cfu ml−1, re-growth was observed
(Fig. 1). At least one out of 8 × 103 bacteria must have
662 U . S CH I LL IN G ER ET A L.
12
10
Log cfu ml–1
8
6
4 cross-resistance to these non-nisin bacteriocins was acquired.
2 who found that spontaneous nisin-resistant mutants of L.
0 monocytogenes ATCC 15131 were still sensitive to all three
0 10 20 30 40 50
Time (h)
Fig. 5 Influence of nisin (100 IU ml−1) on the growth of Listeria
monocytogenes Li3, Lactobacillus sake Lb 706-1a and a mixed culture
of both strains in Standard I Nutrient broth at 30 °C. (), L.
monocytogenes Li3 without nisin; (ž), L. monocytogenes Li3 with
nisin; (T), L. monocytogenes Li3 with Lact. sake Lb 706-1a. (R),
L. monocytogenes Li3 with nisin and Lact. sake Lb 706-1a; (), Lact.
sake Lb 706-1a with L. monocytogenes Li3; (Ž), Lact. sake Lb706-
1a with nisin and L. monocytogenes Li3
survived nisin action and as a consequence, mutants of L.
monocytogenes Scott A resistant to 100 IU of nisin ml−1 can
be expected to occur at a frequency of 10–3–10–4.
Different mechanisms may be responsible for the devel-
opment of resistance. Production of a nisinase seems not to
be involved in the resistance of Listeria to nisin (Mazzotta
and Montville 1997). Alterations in the cell membrane com-
position resulting in the inability of nisin to form pores in the
membrane may contribute to the resistance of spontaneous
mutants of L. monocytogenes (Ming and Daeschel 1993, 1995;
Verheul et al. 1997; Mazzotta and Montville 1997). In a
mutant of L. monocytogenes Scott A resistant to 2000 IU of
nisin ml−1, a higher percentage of straight-chain fatty acids
and a lower percentage of branched-chain fatty acids was
found (Ming and Daeschel 1993), while the amounts of three
different phospholipids in the cytoplasmic membrane were
significantly decreased in this mutant (Ming and Daeschel
1995). Similarily, a mutant of L. monocytogenes Scott A which
was about 12 times more resistant to nisin than the parental
strain contained a reduced amount of diphosphatidylglycerol
in the membrane (Verheul et al. 1997). Studies with a nisin-
resistant mutant of another strain of L. monocytogenes (F6861)
indicated an involvement of the cell wall in the acquisition of
nisin resistance (Davies et al. 1996).
Our results indicate that spontaneous mutants of L. mono-
cytogenes surviving certain concentrations of nisin can be
inhibited by the addition of bacteriocinogenic protective cul-
tures such as the sakacin A producing strain of Lact. sake or
the enterocin B producing Ent. faecium.
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 657–663
A nisin-resistant mutant of L. monocytogenes Scott A which
had been obtained following a single exposure of L. mono-
cytogenes Scott A to 100 IU of nisin ml−1 tolerated nisin
concentrations of 100–500 IU ml−1. This mutant was able to
grow to a high cell density in the presence of 100 IU of nisin
ml−1 and was still sensitive to sakacin A and enterocin B. No
These results agree with those obtained by Rekhif et al. (1994)
different bacteriocins investigated in their study. On the other
hand, Song and Richard (1997) reported that exposure of L.
innocua Lin11 to 20 IU of nisin ml−1 resulted in survivors
that displayed cross-resistance towards nisin, pediocin AcH
and enterococcin EFS2. The nisin-resistant mutant L. mono-
cytogenes Li3 was inhibited by both bacteriocinogenic LAB
under study when grown in mixed culture in Standard I
Nutrient broth. The inhibitory effect of Lact. sake was due
to a combination of pH reduction and the release of large
amounts of sakacin A detectable in the culture supernatant
fluids after different growth periods. A bacteriocin-negative
variant of Lact. sake also had some inhibitory effect on growth
of L. monocytogenes Li3 that can be attributed to the pro-
duction of organic acids and the concomitant pH reduction.
Our results obtained with nisin-resistant derivatives of
Lact. sake Lb 706 and Ent. faecium BFE 900 added to a
culture of L. monocytogenes Scott A in combination with nisin
showed that those non-nisin bacteriocin producing cultures
are in fact able to prevent re-growth of a nisin-resistant
subpopulation of L. monocytogenes. Addition of Lact. sake Lb
706–1a or Ent. faecium BFE 900–6a to L. monocytogenes Scott
A exposed to 100 IU of nisin ml−1 resulted in suppression of
survivors. The bacteriocinogenic strains thus assured a long-
term antilisterial effect.
Inhibition of the nisin-resistant strain of L. monocytogenes
(Li3) by Lact. sake Lb 706–1a and Ent. faecium BFE 900–6a
was also demonstrated in the presence of nisin. Growth of L.
monocytogenes Li3 was hardly affected by the addition of nisin
(100 IU ml−1) but was controlled by both bacteriocinogenic
strains. However, the combination of nisin with sakacin A
produced in situ by Lact. sake Lb 706–1a did not result in
an enhanced inhibitory effect against L. monocytogenes Li3
compared with the sakacin A producer alone.
Our results indicate that a combination of nisin and a
starter or protective culture producing a non-nisin bacteriocin
may be effective in preventing the emergence of spontaneous
nisin-resistant subpopulations of L. monocytogenes. The effec-
tiveness of the bacteriocinogenic LAB may be affected by the
type of strain, its inoculum size, the medium used as well as
pH and temperature. Finally, the behaviour of the protective
cultures in food systems has to be investigated. The influence
of these factors on the inhibition of nisin-resistant L. mono-
cytogenes is currently being studied.

ACKNOWLEDGEMENTS
This study was supported by funds from the European Com-
mission (FAIR-CT96–1148). The authors would like to thank
H. Schäfer for excellent assistance.
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